SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in both pre- and postfusion conformations were determined to average resolutions of 8.7-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass-spectrometry, which revealed highly similar overall processing states of the native glycans to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNP) and its higher-order assemblies were revealed. Overall, these characterizations have revealed the architecture of the SARS-CoV-2 virus in exceptional detail, and shed lights on how the virus packs its ∼30 kb long single-segmented RNA in the ∼80 nm diameter lumen.

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins and the complexes of the spike (S) proteins with the cellular receptor ACE2 or neutralizing antibodies, detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in both pre- and postfusion conformations were determined to average resolutions of 9-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed highly similar overall processing states of the native glycans to that of the recombinant glycoprotein glycans. The in situ architecture of the ribonucleoproteins (RNP) and its higher-order assemblies were revealed. These characterizations have revealed the architecture of the SARS-CoV-2 virus to an unprecedented resolution, and shed lights on how the virus packs its ~30 Kb long single-segmented RNA in the ~80 nm diameter lumen. Overall, the results unveiled the molecular architecture and assembly of the SARS-CoV-2 in native context.

BackgroundAberrant expression of the RON receptor tyrosine kinase is a pathogenic feature and a validated drug target in various types of cancers. Currently, therapeutic antibodies targeting RON for cancer therapy are under intensive evaluation. Here we report the development and validation of a novel humanized anti-RON antibody-drug conjugate for cancer therapy.MethodsAntibody humanization was achieved by grafting sequences of complementarity-determining regions from mouse monoclonal antibody Zt/g4 into human IgG1/κ acceptor frameworks. The selected humanized Zt/g4 subclone H1L3 was conjugated with monomethyl auristatin E using a dipeptide linker to form H-Zt/g4-MMAE. Pharmacokinetic analysis of H-Zt/g4-MMAE was determined using hydrophobic interaction chromatography and a MMAE ADC ELISA kit. Biochemical and biological assays were used for measuring RON expression, internalization, cell viability and death. Therapeutic efficacies of H-Zt/g4-MMAE were validated in vivo using three pancreatic cancer xenograft models. Toxicological activities of H-Zt/g4-MMAE were determined in mouse and cynomolgus monkey.ResultsH-Zt/g4-MMAE had a drug to antibody ratio of 3.77:1 and was highly stable in human plasma with a dissociation rate less than 5% within a 20 day period. H-Zt/g4-MMAE displayed a favorable pharmacokinetic profile in both mouse and cynomolgus monkey. In vitro, H-Zt/g4-MMAE induced RON internalization, which results in killing of pancreatic cancer cells with IC50 values at 10–20 nM. In vivo, H-Zt/g4-MMAE inhibited pancreatic cancer xenograft growth with tumoristatic concentrations at 1~3 mg/kg bodyweight. Significantly, H-Zt/g4-MMAE eradicated tumors across multiple xenograft models regardless their chemoresistant and metastatic statuses. Moreover, H-Zt/g4-MMAE inhibited and eradicated xenografts mediated by pancreatic cancer stem-like cells and by primary cells from patient-derived tumors. Toxicologically, H-Zt/g4-MMAE is well tolerated in mice up to 60 mg/kg. In cynomolgus monkey, H-Zt/g4-MMAE up to 30 mg/kg had a manageable and reversible toxicity profile.ConclusionsH-Zt/g4-MMAE is superior in eradication of pancreatic cancer xenografts with favorable pharmacokinetic profiles and manageable toxicological activities. These findings warrant the transition of H-Zt/g4-MMAE into clinical trials in the future.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0525-0) contains supplementary material, which is available to authorized users.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally with more than 33 million patients diagnosed, taking more than a million lives. Abundant mutations were observed but the functional consequences of these mutations are largely unknown. We report the mutation spectrum, replication dynamics, and infectivity of 11 patient-derived viral isolates in diverse cell lines, including the human lung cancer cell line Calu-3. We observed 46 mutations, including 9 different mutations in the spike gene. Importantly, these viral isolates show significant and consistent variations in replication dynamics and infectivity in tested cell lines, up to a 1500-fold difference in viral titers at 24 h after infecting Calu-3 cells. Moreover, we show that the variations in viral titers among viral isolates are positively correlated with blood clotting function but inversely correlated with the amount of red blood cell and hemoglobin in patients. Therefore, we provide direct evidence that naturally occurring mutations in SARS-CoV-2 can substantially change its replication dynamics and infectivity in diverse human cell lines, with clinical implications in vivo.

Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasia with poor outcome. Currently, the lack of effective therapy has fostered a major effort to discover new targets to treat this malignant cancer. Here we identified the RON receptor tyrosine kinase as a therapeutic target for potential TNBC treatment. We analyzed RON expression in 168 primary TNBC samples via tissue microarray using anti-RON IHC staining and demonstrated that RON was widely expressed in 76.8% TNBC samples with overexpression in 76 cases (45.2%). These results provide the molecular basis to target RON for TNBC therapy. To this end, anti-RON monoclonal antibody Zt/g4-drug monomethyl auristatin E conjugate (Zt/g4-MMAE) was developed with a drug to antibody ratio of 3.29 and tested in a panel of TNBC cell lines with different phenotypes. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulted in cell-cycle arrest followed by massive cell death. The calculated IC 50 values ranged from 0.06 to 3.46 mg/mL dependent on individual TNBC cell lines tested. Zt/g4-MMAE also effectively killed TNBC stem-like cells with RON þ /CD44 þ /CD24 À phenotypes and RON-negative TNBC cells through the bystander effect. In vivo, Zt/g4-MMAE at 10 mg/kg in a Q12 Â 2 regimen completely eradicated TNBC xenografts without the regrowth of xenograft tumors. In conclusion, increased RON expression is a pathogenic feature in primary TNBC samples. Zt/g4-MMAE is highly effective in eradicating TNBC xenografts in preclinical models. These findings lay the foundation for using anti-RON Zt/g4-MMAE in clinical trials as a novel strategy for TNBC treatment. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):

Aberrant expression of the RON receptor tyrosine kinase, a cell surface protein, is a pathogenic feature in pancreatic cancer, which renders it a drug target for targeted therapy. Nevertheless, development of therapeutics targeting RON for pancreatic cancer therapy is hampered due to the lack of full addiction by pancreatic cancer cells to RON signaling for growth and survival. Here we describe a novel strategy using anti-RON antibody-directed drug delivery in the form of an antibody-drug conjugate for inhibition and/or eradication of pancreatic cancers. Monoclonal antibody Zt/g4 specific to the RON Sema domain was selected as the drug carrier based on its ability to induce robust RON internalization. Conjugation of Zt/g4 with monomethyl auristatin E, designated as Zt/g4-MMAE, was achieved through a protease-sensitive dipeptide linker to reach a drug to antibody ratio of 3.29:1. Zt/g4-MMAE was stable in human plasma with a dissociation rate less than 4% within a 10 day period. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulting in cell cycle arrest followed by massive cell death. The maximal effect was seen in pancreatic cancer cells with more than 10 000 receptor molecules per cell. Zt/g4-MMAE also synergized in vitro with chemotherapeutics including gemcitabine, 5-fluorouracil, and oxaliplatin to further reduce PDAC cell viability. In vivo, Zt/g4-MMAE exerts a long-lasting activity, which not only inhibited but also eradicated pancreatic xenograft tumors. These finding indicate that Zt/g4-directed drug delivery is highly effective for eradicating pancreatic tumors. Thus, Zt/g4-MMAE is a novel biotherapeutic with potential for therapy of RON-expressing pancreatic malignancies.

Ebola haemorrhagic fever causes deadly disease in humans and non-human primates resulting from infection with the Ebola virus (EBOV) genus of the family Filoviridae. However, the mechanisms of EBOV lifecycle in host cells, including viral entry, membrane fusion, RNP formation, GP-tetherin interaction, and VP40-inner leaflet association remain poorly understood. This review describes the biological functions of EBOV proteins and their roles in the lifecycle, summarizes the factors related to EBOV proteins or RNA expression throughout the different phases, and reviews advances with regards to the molecular events and mechanisms of the EBOV lifecycle. Furthermore, the review outlines the aspects remain unclear that urgently need to be solved in future research.